How to retrieve sequence information for cry genes

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Bacillus thuringiensis is a gram positive, soil dwelling bacterium which is commonly used as a pesticide. Cry-toxin may be extracted and used as a pesticide. It occurs in the gut of caterpillars of various types of moths and butterflies as like as dark surface of plant. Bacillus thuringiensis was first discovered in 1902 by Japanese biologist "shigetane Ishiwalu" but it was discovered by German scientist "Ernst Berliner" who isolated it as the cause of a disease called schlaffsucht in flour moth. Bacillus thuringiensis is closely related to B.cereus, a soil bacterium and B.anthracis the cause of anthrax, which differ in their plasmids. Upon speculation they palm protenicous endoloning called CRY PROTEINS Which are encoded cry genes. In most strains of Bacillus thuringiensis the cry genes located on plasmid. Cry toxins have specific activities against insect spices of the order of Lepidoptera (moths and butterflies), dipteral (flies and mosquitoes), coleoptera (beetles), hymenoptera (wasps, bees, ants and sawflies) and nematodes. The entomo pathogenic activity of Bt is mainly due to cry toxins. One feature that distinguishes cry proteins is their high specificity for their target insect. More than 200 different cry genes have been isolated. This constitutes an important accept for the control of wide Varity of insect pest. Serves as a important reservoir of cry toxins for production of biological insecticide and insect resistance genetically modified crops. When insect inject toxin crystals, the alkaline pH activates the toxins, this gets inserts. Into the insect midgut epithelial cell forming a pour. This results in cell lysis and iventual death of the insect. A number of receptors in the insect gut have been identified an include amino peptidase N proteins and glycolipids. Spores and crystallise insecticidal protiens produced B thuringiensis have been used to control insect pests. B. thuringiensis based insecticides are often applied as liquid sprays on crop plans, where the insecticide must be injected to be effective.

Advantages in expressing Bt toxins in transgenic crop

The level of toxin expression will be high delivering to sufficient dosage to the pests.

The insects whose toxin expression is within the plant system on the crop perish.

The toxin expression can be modulated by using tissue specific promoters and replaces the use of the synthetic pesticide in the environment.

MATERIALS AND METHOD

BLAST (basic alignment search tool): It is an algorithm for comparing primary biological sequence information, such as the aminoacid sequence of different proteins or the nucleotides of DNA sequences. It can be used to inter functional and evolutionary relationships between sequences as well as help to identify members of gene families.

The Blast program was designed by Eugene ,Myers, Stephen Altschol, Warren Gish, David J.lipam and Webb Miller at NIH and was published in J .Mol.Bio in 1990.

Blast is actually a family of programs :

Nucleotide Blast-Search a nucleotide database using a nucleotide query

Protein Blast-Search protein blast using a protein query

tBlastn -Search nucleotide database using a protein query

tblastx -Search translated nucleotide database using a translated nucleotide query

Blastx-Searches protein database using a translated nucleotide query

Uses of Blast: They can be used for several purposes. These includes Identifying species ,Locating domains ,establishing phylogeney , DNA mapping and comparsion.

FASTA : It is a DNA and Protein sequence alignment software package first described by David J.lipman and Willam R pearson in 1985.It compares a protein sequence to another protein sequence or to a protein database, or a DNA sequence to another DNA sequence or DNA library. FASTA format may be used to represent either single sequences or many sequence in a single step.

Emboss

EMBOSS is a new Open Source software analysis package is specially developed for the molecular biology (e.g. EMBnet) user community. This software automatically copes the data in a variety of formats and allows transparent retrieval of sequence data from the web.

The EMBOSS suite:

This provides a comprehensive set of sequence analysis programs (nearly 150)

Provides a set of core software libraries (AJAX and NUCLEUS)

Integrates other publicly available packages

Encourages the use of EMBOSS in sequence analysis training

Encourages developers elsewhere to use the EMBOSS libraries

Supports all common Unix platforms including Linux, Digital Unix, Irix and Solaris

Protocol 1-To get the sequence retrieval firstly www.ncbi.nlm.nih.gov is typed into the address line of the web browser then select the "Nucleotide" database from the search menu and type in the accession(DQ2416775.1) into the search field and press search to see the record for the above accession. Now click on the CDS link n select FASTA format from the tab to get the sequence of the accession which is then copied and pasted in your notepad and saved as accession DQ2416775.1_cds.

The same procedure is repeated to the accession AY376665.1 to get the sequence which is then saved as AY376665.1_cds.

Protocol 2- To get the sequence alignment the following URL www.ebi.ac.uk/muscle is typed in the address line. Now the CDS sequence of DQ241675.1 and AY376665.1 which is saved in protocol 1 is copied and pasted in any supported format and click Run which will then align the sequences. To retrieve the alignment click on the output file and save the alignment in notepad as file name accessions followed by cds_alignment. To get a new window containing the alignment click start Jalview where the consensus at the bottom is black this is where the Cds sequences aligns.

Protocol 3-The Translation of a nucleotide sequence can be retrieved from the NCBI website. Now to get the sequence type in www.abi.ac.uk/emboss/transeq then copy and paste the CDS for AY376665.1 into the sequence field and click Run. Now copy and paste the output file in notepad and save. (If there is any (*)at the end of the sequence then remove because that indicates the termination codon.) Save the file as AY376665.1_aa

The same procedure is repeated for DQ241675.1 and save the file.

Protocol 4-Homology searching using BLAST(basic local alignment search tool) which provides a method to get rapid search for nucleotide and protein databases .Type the URL www.ncbi.nlm.nih.gov into the address line and click on 'protein' BLAST In the Blast option. Now paste the protein sequence of DQ241675.1_aa and click BLAST to get the result. Each sequence that produced a 'significant' alignment is listed. Now save the BLAST output as a HTML file.

Protocol 5- To get the Multiple Sequence alignment go to http://www.ebi.ac.uk/clustalw2 and paste the FASTA of the cds for DQ241675.1 which is saved from protocol 1 and press enter. Now just copy and paste the FASTA sequence from NCBI. Retrieve the FASTA sequences of M89794 ,Y09787.1 and AY960853.1.Copy and paste the sequences and press run. Now the cladogram underneath the alignment would illustrate the relatedness of the sequences.

RESULTS AND DISCUSSION:

PROTOCOL 1:

Figure 1: FASTA sequence of accession DQ241675.1 by using National centre of biotechnology information

Figure 2: FASTA sequence of accession AY376665.1 by using National centre of biotechnological information

Protocol 2:

Figure 3: The above figure shows the sequence alignment of accessions DQ241675.1 and AY376665.1 by multiple accurate and fast sequence comparsion by log expectation tool

Protocol 3:

Figure 4:The above figure shows the aminoacid sequence of accession AY376665.1

Figure 5: The above figure shows the aminoacid sequence of accession DQ241675.1

Protocol 4:

Figure 6:Homology searching using BLAST from NCBI

Figure 7:

Protocol 5:

Figure 8:

Figure 2:

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